The present invention is in the field of biopesticides. More particularly, this invention relates to the finding that a novel strain of Bacillus pumilus, NRRL accession number B-30087, can inhibit a broad range of fungal plant diseases in vivo. The invention also relates to fungicidal compositions comprising this novel Bacillus strain and the antibiotics and metabolites produced by this strain either alone, or in combination with other chemical and biological pesticides.
For a number of years, it has been known that various microorganisms exhibit biological activity so as to be useful to control plant diseases. Although progress has been made in the field of identifying and developing biological pesticides for controlling various plant diseases of agronomic and horticultural importance, most of the pesticides in use are still synthetic compounds. Many of these chemical fungicides are classified as carcinogens by the Environmental Protection Agency (EPA), are toxic to wildlife and other non-target species. In addition, pathogens may develop resistance to chemical pesticides (see, e.g., Schwinn et al., in: Advances In Plant Pathology: Phytophthora Infestans, The Cause of Late Blight of Potato, p. 244, Academic Press, San Diego, Calif., 1991).
Biological control offers an attractive alternative to synthetic chemical fungicides. Biopesticides (living organisms and the naturally produced compounds produced by these organisms) can be safer, more biodegradable, and less expensive to develop.
Bacilli are known to produce antifungal and antibacterial secondary metabolites (Korzybski et al. (1978) xe2x80x9cSection C: Antibiotics isolated from the genus Bacillus (Bacillaceae)xe2x80x9d in: Antibiotics-Origin, Nature and Properties, American Society for Microbiology, Washington, D.C. (1978) Vol III.) and by Berdy (CRC Handbook of Antibiotic Compounds, Vols. I-XIV, (CRC Press, Inc., Boca Raton, Fla. 1980-87). Compounds known to be produced by B. pumilis include micrococcin P, pumilin, and tetain.
Kawaguchi et al. 1981 (U.S. Pat. No. 4,250,170) isolated a novel water soluble antibiotic from Bacillus with activity against a broad range of gram positive and gram negative bacteria. Stabb et al. (1990) Applied Environ. Microbiol 60:44044412 have identified certain Bacillus spp. (Bacillus spp. includes B. subtilis, B. cereus, B. mycoides, B. thuringiensis) strains that exhibit antifungal activity. These strains have been shown to produce zwittermicin-A and/or kanosamine (Milner et al., (1996) Appl. Environ. Microb. 62:3061-3066), two antibiotic agents that are effective against the soil borne disease damping off, caused by Phytophthora medicaginis, P. nicotianae, P. aphanidermatum or Sclerotinia minor (See Stabb et al., supra). Zwittermicin-A is a water soluble, acid stable linear aminopolyol molecule (see, He et al, (1994) Tetrahedron Lett. 35(16):2499-2502) with broad spectrum activity against many fungal and bacterial plant pathogens. Kanosamine (Milner et al., 1996) also inhibits a broad range of fungal plant pathogens and a few bacterial species.
U.S. Pat. No. 5,049,379 to Handelsman et al. describes how Zwittermicin-A producing B. cereus control damping off in alfalfa and soybeans. When the seed was coated with B. cereus ATCC 53522, the pathogenic activity of root rot fungus was inhibited. Similarly, application of spore-based formulations of certain B. cereus strains to soybean seeds or the soil surrounding the seeds has been shown to improve soybean yield at field sites. (See, Osburne et al. (1995) Am. Phytopathol. Soc. 79(6):551-556). Methods of applying biopesticides are well known in the art and include, for example, wettable powders, dry flowables, microencapsulation, and liquid formulations of the microbe, whole broth or antibiotic fractions from suitable cultures. (See e.g., U.S. Pat. No. 5,061,495 to Rossall or U.S. Pat. No. 5,049,379 to Handelsman).
Tsuno et al. (Takashi Tsuno, Chiharo Ikeda, Kei-ichi Numata, Koju Tomita, Masataka Konishi and Hiroshi Kawaguchi (1986) J. Antibiotics XXXIX(7):1001-1003) report on a new amino sugar antibiotic from B. pumilus with activity against a broad range of bacteria in vitro.
Leifert et al., J. Appl. Bacteriol. 78:97-108 (1995), reported the production of anti-Botrytis and anti-Alternaria antibiotics by two Bacillus strains, B. subtilis CL27 and B. pumilis CL 45. The whole broth and cell-free filtrates were active against Botrytis and Alternaria in in vitro tests and were active against Botrytis in in vivo small plant tests on Astilbe. Leifert et al. (1997) U.S. Pat. No. 5,597,565 disclose B. subtilis, B. pumilis, and B. polymyxa that are particularly effective at inhibiting post harvest disease causing fungi, Alternaria brassicicola and Botrytis cinerea. They also disclose the presence of antibiotics produced in the cell-free culture filtrate and their activity at different pH values, but they do not identify these compounds. The compounds from B. subtilis lose activity at low pH, while the activity from the B. pumilus extracts occurs only at pH values below 5.6. Leifert et al. (1998) U.S. Pat. No. 5,780,080 discloses cabbages that can be treated with B subtilis, B pumilis, and B. polymyxa strains to inhibit Alternaria brassicicola and Botrytis cinerea. 
Loeffler et al. (1986) J. Phytopathology 115:204-213, disclose B. subtilis, B. pumilus, B. licheniformis, and B. coagulans strains that produce various antibiotics with antifungal and antibacterial activity. B. pumilus produced bacilysin and iturin A. Bacilysin is a very small compound with a molecular weight of 270, that inhibits only yeast. The iturins, which are soluble in polar solvents, have broad antifungal and antibacterial activity.
Rossall (1994) U.S. Pat. No. 5,344,647 discloses Bacillus subtilis strains with broad anti-fungal activity. Rossall""s (1991) U.S. Pat. No. 5,061,495 provides a novel antibiotic from B. subtilis that is 63,500 Dalton, precipitates at a pH below 5 and has activity against gram positive bacteria and fungi (Botrytis and Erysiphe). Sholberg et al. (1995) Can. J. Microbiol. 41:247-252, Swinburne et al. (1975) Trans. Brit. Mycol. Soc. 65:211-217, Singh and Deverall, (1984) Trans. Br. Mycol. Soc. 83:487-490, Ferreira et al. (1991) Phytopathology 81:283-287 and Baker et al. (1983) Phytopathology 73:1148-1152. All disclose the use of Bacillus spp. and Bacillus subtilis as biocontrol agents of fungal plant pathogens. Pusey et al. (1988) Plant Dis. 72:622-626, Pusey and Robins (U.S. Pat. No. 5,047,239), and McKeen et al (1986) Phytopathology 76:136-139 disclose control of post harvest fruit rot using B. subtilis. McKeen et al, supra, have shown that antibiotics similar to the low molecular weight iturin cyclic polypeptides contribute to this fungicidal activity of B. subtilis. 
Liu et al. (1995) U.S. Pat. No. 5,403,583 disclose a Bacillus sp., ATCC 55000 and a method to control the fungal plant pathogen, Rhizoctonia solani. Islam and Nandi (1985) J. Plant Dis. Protect 92(3):241-246, disclose a Bacillus sp. with antagonism to Drechslera oryzae, the causal agent of rice brown spot. The same authors, Islam and Nandi (1985) J. Plant Dis. Protect. 92(3):233-240, also disclose in-vitro antagonism of Bacillus sp. against Drechslera oryzae, Alternaria alternata and Fusarium roseum. They discuss three components in the culture filtrate. The most active antibiotic was highly soluble in water and methanol with a UV peak at 255 nm and a shoulder at 260 um, that proved to be a polyoxin-like lipopeptide. Cook (1987) Proceedings Beltwide Cotton Production-Mechanization Research Conference, Cotton Council, Memphis, pp. 43-45 discloses the use of a suspension of Bacillus sp. to reduce the number of cotton plants killed by Phymatotrichum omnivorum, a cause of cotton root rot.
B""Chir and Namouchi (1988) (Revue Nematologique 11(2):263-266) report on a Bacillus pumilus that stimulates nematode trapping fungi to increase their ability to trap nematodes. B""Chir and Belkadhi (1986) (Med. Fac. Landbouww. Rijksuniv. Gent 51/3b:1295-1310) discuss the cellular interactions of a fungus (Fusarium) and nematodes that cause infection in citrus. The fungus is associated with B. pumilis (they occur together) and when the nematode is also there, the fungus is more severe. B. pumilus appears to be providing food for the nematodes. Gokte and Swarup (1988) (Indian J. Nematol. 18(2):313-318) report on B. pumilus that are nematicidal, but they do not report any antifungal activity. Slabospitskaya et al. (1992) (Mikrobiol Zh (Kiev) 54(6):16-22) compare many different Bacillus, including B. pumilus for their ability to produce chitinases, but they report no activity on plant pathogens. The B. pumilus produce the lowest chitinase levels. McInroy et al. (1995) Plant and Soil 173(2):337-342, did a survey of the many types of bacteria, including many Bacillus and B. pumilus that are endophytes within plant stems and roots. However, they show no evidence that these endophytic strains are antifungal. Chernin et al. (1995) Molecular Genetics, found a Bacillus pumilus that has a wide spectrum of activity against bacteria (e.g., Xanthomonas, Pseudomonas, Erwinia) and fungi that cause plant disease. Fey et al. (1991) Akad Landwirts Kart, report on B. pumilus strains that provide seed potatoes some protection from Rhizoctonia solani. 
A novel antibiotic-producing Bacillus sp. is provided that exhibits antifungal activity only on certain specific plant pathogens and no antibacterial activity. Also provided is a method of treating or protecting plants, fruit and roots from fungal infections comprising the step of applying an effective amount of an antibiotic-producing Bacillus sp. The antibiotic-producing Bacillus sp. can be provided as a suspension in a whole broth culture or as an antibiotic-containing supernatant obtained from a whole broth culture of an antibiotic-producing Bacillus sp. Also provided is a novel water-soluble antibiotic that exhibits specific antifungal activity and no antibacterial activity.
The present invention provides a biologically pure culture of a strain having all the identifying characteristics of a novel strain of Bacillus sp. or mutants thereof with antifungal activity only on specific plant pathogens such as rusts, powdery mildews and downy mildews. This novel strain is deposited with the NRRL on Jan. 14, 1999 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure under Accession No. B-30087. The invention also includes methods of preventing and treating fungal diseases in plants, including plant roots, using such bacterial strains or antibiotic-containing supernatants or pure antibiotics obtained from such bacterial strains. The invention also includes a water soluble antifungal antibiotic with a molecular weight of less than 10,000 Dalton, slightly heat labile, positively charged, and an HPLC peak with UV absorbance at a maximum of 280 nm and a shoulder at 230 nm.
Definitions
As used in the specification and claims, the singular form xe2x80x9caxe2x80x9d, xe2x80x9canxe2x80x9d and xe2x80x9cthexe2x80x9d include plural references unless the context clearly dictates otherwise. For example, the term xe2x80x9ca cellxe2x80x9d includes a plurality of cells, including mixtures thereof.
As used herein, the term xe2x80x9ccomprisingxe2x80x9d is intended to mean that the compositions and methods include the recited elements, but not excluding others. xe2x80x9cConsisting essentially ofxe2x80x9d when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. xe2x80x9cConsisting ofxe2x80x9d shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
The term xe2x80x9cisolatedxe2x80x9d is used interchangeably with xe2x80x9cbiologically purexe2x80x9d and means separated from constituents, cellular and otherwise, in which the strain or metabolite are normally associated with in nature.
As used herein, xe2x80x9cbiological controlxe2x80x9d is defined as control of a pathogen or insect by the use of a second organism. Known mechanisms of biological control include enteric bacteria that control root rot by out-competing fungi for space on the surface of the root. Bacterial toxins, such as antibiotics, have been used to control pathogens. The toxin can be isolated and applied directly to the plant or the bacterial species may administered so it produces the toxin in situ.
The term xe2x80x9cfungusxe2x80x9d or xe2x80x9cfungixe2x80x9d includes a wide variety of nucleated spore-bearing organisms that are devoid of chlorophyll. Examples of fungi include yeast, molds, mildews, rusts, and mushrooms.
The term xe2x80x9cbacteriaxe2x80x9d includes any prokaryotic organism that does not have a distinct nucleus.
xe2x80x9cFungicidalxe2x80x9d means the ability of a substance to increase mortality or inhibit the growth rate of fungi.
xe2x80x9cAntibioticxe2x80x9d includes any substance that is able to kill or inhibit a microorganism. Antibiotics may be produced by a microorganism or by a synthetic process or semisynthetic process. The term, therefore, includes a substance that inhibits or kills fungi for example, zwittermicin-A or kanosamine,
xe2x80x9cAntifungalxe2x80x9d includes any substance that is able to kill or inhibit the growth of fungi.
The term xe2x80x9cculturingxe2x80x9d refers to the propagation of organisms on or in media of various kinds.
xe2x80x9cWhole broth culturexe2x80x9d refers to a liquid culture containing both cells and media.
xe2x80x9cSupernatantxe2x80x9d refers to the liquid broth remaining when cells grown in broth are removed by centrifugation, filtration, sedimentation, or other means well known in the art.
An xe2x80x9ceffective amountxe2x80x9d is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations. In terms of treatment and protection, an xe2x80x9ceffective amountxe2x80x9d is that amount sufficient to ameliorate, stabilize, reverse, slow or delay progression of the fungal or bacterial disease states.
xe2x80x9cPositive controlxe2x80x9d means a compound known to have pesticidal activity. xe2x80x9cPositive controlsxe2x80x9d include, but are not limited to, commercially available chemical pesticides. The term xe2x80x9cnegative controlxe2x80x9d means a compound known not to have pesticidal activity. Examples of negative controls are water or ethyl acetate.
The term xe2x80x9csolventxe2x80x9d includes any liquid that holds another substance in solution. xe2x80x9cSolvent extractablexe2x80x9d refers to any compound that dissolves in a solvent and which then may be isolated from the solvent. Examples of solvents include, but are not limited to, organic solvents like ethyl acetate.
The term xe2x80x9cmetabolitexe2x80x9d refers to any compound, substance or byproduct of a fermentation of a microorganism that has pesticidal activity. Antibiotic as defined above is a metabolite specifically active against a microorganism.
A xe2x80x9ccompositionxe2x80x9d is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
We describe a biologically pure culture of a strain having all the identifying characteristics of a novel antibiotic-producing strain of Bacillus sp. NRRL No. B-30087, and mutants thereof, that have antifungal activity only on specific plant pathogens and no antibacterial activity. In one aspect, the strain is Bacillus pumilis deposited under NRRL No. B-30087, and mutants of the strain.
In other aspects, the strain is a variant of NRRL No. B-30087 which has all the identifying characteristics (as provided below) of NRRL No. B-30087. A variant may also be identified as having a genome that hybridizes under conditions of high stringency to the genome of NRRL No. B-30087. xe2x80x9cHybridizationxe2x80x9d refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. Hybridization reactions can be performed under conditions of different xe2x80x9cstringency.xe2x80x9d In general, a low stringency hybridization reaction is carried out at about 40xc2x0 C. in 10xc3x97SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50xc2x0 C. in 6xc3x97SSC, and a high stringency hybridization reaction is generally performed at about 60xc2x0 C. in 1xc3x97SSC.
A variant of NRRL No. B-30087 may also be defined as a stain having a genomic sequence that is greater than 85%, more preferably greater than 90% or more preferably greater than 95% sequence identity to the genome of NRRL No. B-30087. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of xe2x80x9csequence identityxe2x80x9d to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.
This invention further provides the supernatants obtained from the above noted cultures. The supernatant may be obtained by methods well known in the art including: centrifugation; filtration; sedimentation; and the like.
In another aspect, the invention encompasses an isolated metabolite that is a water-soluble antifungal antibiotic. The metabolite is isolated from the strains of this invention and described above. It has the characteristics of being less than 10,000 Dalton, UV absorption peak at 280 nm and shoulder at 230 nm, acid and base stable, slightly heat labile over 80xc2x0 C., and positively charged with activity on specific plant pathogens, but with no activity on bacteria. This invention further provides a process for producing this metabolite, the method comprising culturing a strain of this invention and isolating the active metabolite using the methods described below.
Further provided by this invention are compositions comprising any of the above strains (including mutants or variants thereof), supernatants, and metabolites, alone or in combination with each other, and a carrier. These compositions may be further supplemented by the addition of at least one chemical or biological pesticide. These compositions may take the form of various formulations, which include, but are not limited to, a wettable powder, a granule formulation, an aqueous suspension, an emulsifiable concentrate or microencapsulation.
In order to achieve good dispersion and adhesion of compositions within the present invention, it may be advantageous to formulate the whole broth culture, supernatant and/or metabolite/antibiotic with components that aid dispersion and adhesion. Accordingly, suitable formulations will be known to those skilled in the art (wettable powders, granules and the like, or can be microencapsulated in a suitable medium and the like, liquids such as aqueous flowables and aqueous suspensions, and emulsifiable concentrates). Other suitable formulations will be known to those skilled in the art.
Any of the above noted strains, metabolites, supernatants and compositions containing these active ingredients, may be used to provide a method of treating or protecting plants, roots or fruit from fungal infections. The method comprises applying an effective amount of a strain, metabolite, supernatant or compositions containing these active ingredients, alone or in combination with each other and/or another biologic or chemical pesticide, to the infected root, plant or fruit. Effective amounts of these compositons also can be applied to a plant, root or fruit to prevent such infestations.
In further aspect, the invention encompasses a method of treating or protecting plants, roots or fruit from fungal diseases comprising applying an effective amount of the antibiotic produced by a strain having all the identifying characteristics of the novel strain Bacillus sp. NRRL No. B-30087. In one embodiment, the strain is Bacillus sp. NRRL No. B-30087.
Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.
The following examples are intended to illustrate, but not limit the invention.